Iron Rescue of Deferoxamine Toxicity in human RPE cells

Abstract

Purpose: Deferoxamine (DFX) is an iron chelator commonly used to treat iron overload in patients requiring regular blood transfusions. Several ocular complications have been reported, while in vitro studies have confirmed its toxicity in hepatic, cortical brain cells and bovine RPE cells. Our study is the first to examine DFX’s toxicity in an established human RPE cell line (ARPE19) and whether apoptosis, necrosis, or the chelation of minerals plays a role in toxicity.

Methods: ARPE19 cells were cultured on 96 well plates. MTT assays and TUNEL staining were used to assess cell survival. To investigate mechanisms of cell death, inhibitors of necroptosis (Nec-1) and apoptosis (z-Vad) were added to DFX treated cells. Additional plates were incubated with ZnCl2 or FeCl with and without DFX.

Results: DFX toxicity to ARPE19 cells was confirmed by an MTT assay, and verified by TUNEL staining. No toxicity was observed at 24 hours, but decreased cell survival began at day 2 and progressed through day 5. There was no significant difference in cell survival between 0.2, 0.4, and 0.8 mg/mL of DFX. While investigating the role of necroptosis and apoptosis in DFX-induced toxicity, we found that neither Nec-1 nor Z-Vad improved cell survival at days 2 and 3. We then focused on whether the chelation of minerals could reduce DFX’s toxicity, but first checked for direct toxicity of iron and zinc. Zinc alone showed a dose dependent response with high doses (0.04, 0.08, and 0.16M) demonstrating significant toxicity while lower doses (10, 20, 40 uM) had no effect on cell survival. Iron alone showed no toxicity at concentrations of 0.05, 0.1 and 0.2 M. Rescue of DFX toxicity was then tested by adding zinc and iron to 0.4 mg/mL DFX treated RPE cells. The addition of zinc did not improve cell survival. However, iron was able to rescue DFX toxicity when used at ratios of 1:1 and 2:1 (Fe:DFX). A favorable effect was also noted when iron was premixed with DFX two hours before adding to cell cultures. However, a two day delay in the addition of iron to DFX treated cells was unable to prevent toxicity.

Conclusions: We are the first to confirm DFX toxicity in a human RPE cell line. DFX toxicity appears to be time dependent. We found no role for necroptosis or apoptosis in cell death. However, chelation with iron at specific concentrations rescues RPE cells from DFX toxicity.

Keywords: 503 drug toxicity/drug effects • 701 retinal pigment epithelium